PPAR-γ基因启动子甲基化及表达水平对慢加急性乙型肝炎肝衰竭患者预后评价

发布时间：2018-06-02 01:28

本文选题：慢加急性乙型肝炎肝衰竭 + 过氧化物酶体增殖激活受体γ　；参考：《山东大学》2016年硕士论文

【摘要】：研究背景与目的慢加急性乙型肝炎肝衰竭(acute-on-chronic hepatitis B liver failure, ACHBLF)病死率高,严重危害人类健康。最近的研究表明,促炎细胞因子在ACHBLF的发生和发展过程中发挥着十分重要的作用。有研究证明,过氧化物酶体增殖物激活受体y(peroxisome proliferator-activated receptor gamma, PPAR-γ)参与抗炎反应,但其在ACHBLF中发挥的作用仍不明确。因此,本研究旨在通过检测PPAR-γ基因启动子甲基化及表达水平,揭示PPAR-γ在ACHBLF发生发展中的作用及其预测ACHBLF短期预后的潜在临床应用价值。研究方法本研究共纳入了161例受试者,包括81例ACHBLF患者,50例慢性乙型肝炎(chronic hepatitis B, CHB)患者,以及30例健康对照者。用实时荧光定量PCR (quantitative real-time PCR)法检测外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)中PPAR-γ的mRNA水平；用甲基化特异性PCR(methylation specific PCR, MSP)检测PPAR-γ基因启动子甲基化情况；用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测血浆中TNF-α和IL-6的水平。应用SPSS V17.0软件进行统计学相关分析,两组之间差异比较采用两独立样本t检验,Mann-Whitney U检验或卡方检验,PPAR-γ表达水平与临床资料的相关性分析采用Spearman相关分析。P0.05认为有统计学意义。结果1.ACHBLF患者PPAR-γ mRNA水平与CHB患者相比显著升高(Z=-4.003,P0.001)。然而,ACHBLF患者PPAR-γ mRNA水平仍显著低于健康对照组(Z=-4.632,P0.001)。同时,CHB患者中PPAR-γ表达水平显著低于健康对照组(Z=-6.037,P0.001)。2.对ACHBLF患者PPAR-γ mRNA水平与临床指标进行线性相关分析结果显示,PPAR-γ的表达水平与总胆红素(total bilirubin, TBIL)(r=-0.280,P=0.011)及国际标准化比值(international normalized ratio,INR)(r=-0.230,P=0.039)呈显著负相关。ACHBLF患者PPAR-γ mRNA水平与谷丙转氨酶(alanine aminotranferase,ALT).血肌酐(creatinine,Cr)及乙型肝炎病毒DNA(hepatitis B virUs-DNA,HBV-DNA)载量之间未发现显著相关关系。3.ACHBLF患者中PPAR-γ启动子的甲基化率比CHB患者显著降低(CpG-1,x2=8.918,P=0.003；CpG-2,x2=9.268,P=0.002),而仍然高于健康对照组(CpG-1,x2=6.691,P=0.009；CpG.2,x2=5.050,P=0.025).同时,CHB患者两个CpG岛的甲基化率均明显高于健康对照组(CpG-1,X2= 21.333,P0.001；CpG-2,x2=19.448,P0.001).4.对所有受试者甲基化组与非甲基化组的PPAR-γ mRNA水平进行比较。结果显示,在CpG-1或CpG-2发生甲基化的情况下,PPAR-γ的相对表达量将显著减少(CpG-1,Z=-6.613,P0.001；CpG-2,Z=-7.712, P0.001),并且当CpG-1及CpG-2同时发生甲基化时对PPAR-γ的表达抑制效果增强(Z=-4.085,P0.001)。5.受试者的血浆TNF-α和IL-6水平通过酶联免疫吸附试验测定。ACHBLF患者中两种细胞因子水平均显著高于CHB患者(TNF-α, t=6.649,P0.001；IL一6,t=6.784,P0.001)和健康对照组(TNFα,t=5.582, P0.001；IL-6,t=5.961,P0.001).ACHBLF患者中甲基化组的血浆细胞因子水平显著高于非甲基化组(TNF-α,t=2.312,P=0.023；IL-6,t=2.639, P=0.012).6.ACHBLF患者3个月的死亡率为54.32%(44/81),平均生存时间为53.47天(SE 4.057,95％CI 45.39-61.54).存活组的PPAR-γ mRNA水平显著高于死亡组(Z=-3.489,P0.001)。ACHBLF患者甲基化组的预后显著差于非甲基化组(χ2=11.140,P0.001)。ACHBLF存活组PPAR-γ表达水平随时间延长而显著增加,而死亡组维持于较低水平。我们评估了PPAR-γ mRNA水平、PPAR-γ启动子甲基化对预测ACHBLF短期预后的价值。ROC曲线下面积分别为0.726(SE 0.059,95% CI 0.611-0.841), 0.657(SE 0.062,95％CI 0.535-1.778).结论ACHBLF患者与CHB患者相比PPAR-γ表达水平升高,启动子甲基化率降低。PPAR-γ的表达水平及启动子甲基化状态与ACHBLF的严重程度和预后密切相关。并且,PPAR-γ可能通过下调TNF-α和IL-6,对于改善ACHBLF预后具有潜在意义,PPAR-γ的启动子甲基化状态及表达水平对于预测ACHBLF的短期预后具有重要价值。
[Abstract]:Research background and objective acute-on-chronic hepatitis B liver failure (ACHBLF) has high mortality and serious harm to human health. Recent studies have shown that proinflammatory cytokines play a very important role in the development and development of ACHBLF. Studies have shown that peroxisome proliferation is stimulated. The active receptor y (peroxisome proliferator-activated receptor gamma, PPAR- gamma) is involved in anti-inflammatory reactions, but its role in ACHBLF is still not clear. Therefore, this study aims to reveal the role of PPAR- gamma in the development of ACHBLF and to predict the potential of ACHBLF and short-term prognosis by detecting the methylation and expression level of PPAR- gamma promoter. This study included 161 subjects including 81 ACHBLF patients, 50 patients with chronic hepatitis B (chronic hepatitis B, CHB), and 30 healthy controls. The detection of peripheral blood mononuclear cells by real-time fluorescent quantitative PCR (quantitative real-time PCR) (peripheral blood mononuclear) The mRNA level of PPAR- gamma in PBMCs; the methylation specific PCR (methylation specific PCR, MSP) was used to detect the methylation of the promoter of the PPAR- gamma gene; the enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay) was used to detect the level of the plasma in the plasma. The two groups were used for statistical analysis. The difference was compared with two independent samples t test, Mann-Whitney U test or chi square test. The correlation analysis of PPAR- gamma expression level and clinical data was statistically significant by Spearman correlation analysis.P0.05. Results the level of PPAR- y mRNA in 1.ACHBLF patients was significantly higher than that of CHB patients (Z=-4.003, P0.001). The level of AR- gamma mRNA was still significantly lower than that in the healthy control group (Z=-4.632, P0.001). At the same time, the expression level of PPAR- gamma in CHB patients was significantly lower than that in the healthy control group (Z=-6.037, P0.001).2. (Z=-6.037, P0.001).2.. .280, P=0.011) and international standardized ratio (international normalized ratio, INR) (r=-0.230, P=0.039) showed a significant negative correlation with PPAR- gamma mRNA level and alanine transaminase (alanine). There was no significant phase between the serum creatinine and hepatitis B virus. The methylation rate of PPAR- gamma promoter in.3.ACHBLF patients was significantly lower than that of patients with CHB (CpG-1, x2=8.918, P=0.003, CpG-2, x2=9.268, P=0.002), but still higher than that of the healthy control group (CpG-1, x2=6.691, P=0.009. 333, P0.001, CpG-2, x2=19.448, P0.001).4. compared the PPAR- gamma mRNA levels between the methylation and the non methylation groups of all the subjects. The results showed that the relative expression of PPAR- gamma would be significantly reduced when CpG-1 or CpG-2 occurred methylation. The inhibitory effect of methylation on the expression of PPAR- gamma enhanced (Z=-4.085, P0.001).5. subjects' plasma TNF- alpha and IL-6 levels were significantly higher than those of CHB patients (TNF- alpha, t=6.649, P0.001, IL a 6,.5.) and healthy control group by enzyme linked immunosorbent assay (ELISA). The plasma cytokine levels in the methylation group of the patients with IL-6, t=5.961, P0.001) were significantly higher than those in the non methylation group (TNF-, t=2.312, P=0.023; IL-6, t=2.639, P=0.012), and the mortality of 3 months was 54.32% (44/81) and the average survival time was 53.47 days. The prognosis of the methylation group in the patients with higher than the death group (Z=-3.489, P0.001) was significantly worse than that in the non methylation group (x 2=11.140, P0.001).ACHBLF survival group PPAR- gamma expression level increased significantly with time, while the death group remained at a lower level. We evaluated PPAR- gamma mRNA level, PPAR- gamma promoter methylation for ACHBLF short-term PREPREDICTION. The area under the post value.ROC curve was 0.726 (SE 0.059,95% CI 0.611-0.841) and 0.657 (SE 0.062,95%CI 0.535-1.778). Conclusion ACHBLF patients were higher in PPAR- gamma expression compared with those of CHB patients. Starting Zi Jiaji rate decreased the expression level of.PPAR- gamma and the state of promoter methylation was closely related to the severity and prognosis of CHB. Moreover, PPAR- gamma may be of potential significance for improving the prognosis of ACHBLF by down regulation of TNF- alpha and IL-6. The methylation status and expression level of PPAR- gamma are of great value for predicting the short-term prognosis of ACHBLF.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.3;R512.62

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